1. Field of the Invention
The present invention relates to a process for the production, or regeneration, of nucleoside 5'-triphosphate (NTPs) from nucleoside 5'-diphosphates (NDPs) other than adenosine 5'-diphosphate (ADP), and to applications of the process to, for example, the synthesis of oligosaccharides.
2. Description of the Related Art
Development of the nucleic acid industry, in forms such as nucleic acid fermentation or enzymatic degradation of nucleic acid, has enabled low cost manufacture of nucleosides and nucleoside 5'-monophosphates (NMPs), some of which are produced and sold as pharmaceuticals or as raw materials for pharmaceuticals. Also, development of pharmaceuticals making use of nucleosides, nucleotldes, and their derivatives has been actively performed. Moreover, recent advancement of sugar chain engineering entails active studies of synthesis of sugar nucleotides, which serve as substrates in enzymatic synthesis of oligosaccharides.
Thus, in contrast to the case of NMPs, which are manufactured and supplied economically, no economical manufacturing method for NTPs, led by adenosine 5'-triphosphate, has so far been established, although there have been known a number of synthesis methods, including chemical synthesis methods and methods making use of a microorganism or an enzyme, and therefore, NTPs currently available on the market are quite expensive.
Recently, many studies have been conducted to develop techniques for the synthesis of oligosaccharides by use of glycosyltransferase and making use of sugar nucleotide as a substrate. Of such studies, a sugar nucleotide recycling method proposed by Scripps Research Institute in the U.S.A. has become of interest (PCT Kohyo Publication No. 7-500248 and JP-A No. 7-79792). This method employs NTP and sugar 1-phosphate as substrates and also uses sugar nucleotide pyrophosphorylase and glycosyltransferase, to thereby synthesize a sugar nucleotide, and simultaneously, while the resultant sugar nucleotide is used as a monosaccharide donor, glycosyltransfer reaction is performed efficiently, to thereby synthesize an oligosaccharide. This method is characterized in that the NDP produced from the glycosyltransfer reaction is regenerated as NTP by the mediation of a combination of phosphoenolpyruvic acid and pyruvate kinase, so that the NTP is reused as a substrate for the synthesis of sugar nucleotide, to thereby reduce the amount of expensive NTP and make the glycosyltransfer reaction efficient, resulting in an expected reduction in costs for the manufacture of oligosaccharides.
The above-mentioned recycling method, though advantageous in that it does not use a large amount of expensive NTP, requires a large amount of expensive phosphoenolpyruvic acid for regenerating NTP. Therefore, this method is not necessarily satisfactory in practice.
In the conversion reaction from NDP to NTP, other enzymes, e.g., nucleoside-diphosphate kinase, may be used in place of pyruvate kinase. However, in this: case also, expensive adenosine 5'-triphosphate (ATP) is required as a phosphate donor, and therefore, such a replacement does not effect a radical resolution.
Accordingly, the present invention is directed to a more practical method for the synthesis, or regeneration, of NTP from NDP without use of expensive phosphoenolpyruvic acid or ATP, and also to applications of the method in, for example, the synthesis of oligosaccharides.
The present inventors have conducted careful studies in an attempt to attain the above object, and have found that conventionally known polyphosphate kinase (Biochim. biophys. Acta., 26, 294-300 (1957)) is endowed with activity capable of phosphorylating NDPs other than ADP, through use of polyphosphate as a phosphate donor, to thereby synthesize NTPs. The inventors have extended their studies toward applications of this method to the synthesis of oligosaccharides, and as a result, they were able to confirm that the method is more practical than the aforementioned sugar nucleotide recycling method. The present invention was accomplished based on these findings.